title: An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues.
abstract: Formalin is the most commonly used fixative for light microscopy because of its preservation of -morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of -formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97(o)C in a water bath) for 40 min in 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris-EDTA-SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2-40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists.
authors.affiliation: Immunopathology Laboratory, Department of Pathology, The University of Iowa, Iowa City, IA, USA. sergei-syrbu@uiowa.edu
authors.forename: Sergei I
authors.initials: SI
authors.lastname: Syrbu
authors.forename: Michael B
authors.initials: MB
authors.lastname: Cohen
mesh_terms: D000818:Animals; D000941:Antigens / Q000737:chemistry*; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D006361:Heating; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D009369:Neoplasms / Q000473:pathology; D016612:Paraffin Embedding / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000941:Antigens; D005404:Fixatives; D005557:Formaldehyde